Podosomes are highly dynamic actin-rich adhesion structures in cells of myeloid lineage and some transformed cells. Unlike transformed mesenchymal cell types, podosomes are the sole adhesion structure in macrophage and thus mediate all contact with adhesion substrate, including movement through complex tissues for immune surveillance. The existence of podosomes in inflammatory macrophages and transformed cell types suggest an important role in tissue invasion. The proteome, assembly, and maintenance of podosomes are emerging, but remain incompletely defined. Previously, we reported a formin homology sequence and actin assembly activity in association with macrophage β-3 integrin. In this study we demonstrate by quantitative RT-PCR and Western blotting that the formin FRL1 is specifically upregulated during monocyte differentiation to macrophages. We show that the formin FRL1 localizes to the actin-rich cores of primary macrophage podosomes. FRL1 co-precipitates with beta-3 integrin and both fixed and live cell fluorescence microscopy show that endogenous and overexpressed FRL1 selectively localize to macrophage podosomes. Targeted disruption of FRL1 by siRNA results in reduced cell adhesion and disruption of podosome dynamics. Our data suggest that FRL1 is responsible for modifying actin at the macrophage podosome and may be involved in actin cytoskeleton dynamics during adhesion and migration within tissues.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of pro-inflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display co-expression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-κB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-κB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-κB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-κB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

Formins are actin binding proteins conserved across species from plants to humans. The formin family is defined by their common FH2 domains. The 15 distinct human formins are involved in a broad range of cellular functions, including cell adhesion, cytokinesis, cell polarity, and cell morphogenesis. Their commonality is actin polymerization activity inherent to FH2 domains. While still requiring much study, biochemical activity of formins has been carefully described. In contrast, much less is known of their activities in complex living systems. With the diversity of the formin family and the actin structures that they affect, an extensive future of study beckons. In this study, we report the expression level of all 15 formins in 22 different human cell and tissue types using quantitative real-time PCR (qPCR). Identification of major themes in formin expression and documentation of expression profiles should facilitate the cellular study of formins.

These results suggest that adhesion complexes actively promote actin assembly from their cytosolic face in order to establish a mechanical linkage with the extracellular matrix.